E. Single-stranded m13 dna isolation using phenol

This isolation procedure (23) is the method of choice for preparation of M13-based templates to be used in Sequenase[TM] catalyzed dye-terminator reactions. A pre-incubated early log phase JM101 culture is prepared by transferring a thawed glycerol stock into 50 ml of liquid media and incubating for 1 hour at 37degC with no shaking. M13 plaques are picked with a sterile toothpick and placed into 1.5 ml aliquots of the early log phase JM101 culture, which are incubated in a 37deg C shaker for 4-6 hours. After incubation, the bacterial cells are pelleted by centrifugation and the viral containing supernatant is transferred to a clean tube. The phage particle are precipitated with PEG, collected by centrifugation, and the pellet is resuspended in buffer. The phage protein coat is denatured and removed by one phenol and two ether extractions. After ethanol precipitation, the dried DNA pellet is resuspended in buffer, and the concentration and purity crudely are assessed by agarose gel electrophoresis against known standards.

Protocol

1. Prepare an early log phase culture of JM101, as above, and pick M13-based plaques with sterile toothpicks into 12 X 75 mm Falcon tubes containing 1.5 ml aliquots of the cells. Incubate for 4-6 hours at 37degC with shaking at 250 rpm.

2. Transfer the culture to 1.5 ml microcentrifuge tubes and centrifuge for 15 minutes at 12,000 rpm at 4degC.

3. Pipette the top 1 ml of supernatant to a fresh 1.5 ml microcentrifuge tube containing 0.2 ml 20% PEG/2.5 M NaCl to precipitate the phage particles. Mix by inverting several times and incubate for 15-30 minutes at room temperature.

4. Centrifuge for 15 minutes at 12,000 rpm at 4degC to collect the precipitated phage. Decant the supernatant and remove residual PEG supernatant by suctioning twice.

5. Resuspend the pellet in 100 ul of 10 mM Tris-HCl, pH 7.6 by vortexing, and add 50 ul of TE-saturated phenol.

6. Extract the DNA with phenol and twice with ether, as discussed above, and then ethanol precipitate.

7. Resuspend the dried DNA in 6 ul of 10:0.1 TE for use in single-stranded Sequenase[TM] catalyzed dye-terminator sequencing reactions.

F. Biomek-automated modified-Eperon isolation procedure for single-stranded m13 dna

This semi-automated method is a modification of a previously reported procedure (24,25), and allowed the simultaneous isolation of 48 single-stranded DNAs per Biomek 1000 robotic workstation within 3 hours (26). Basically, M13 plaques are picked with sterile toothpicks into aliquots of early log phase JM101, prepared as discussed above. The phage infected cultures are incubated in a 37degC shaker for 4-6 hours, transferred into microcentrifuge tubes, centrifuged to separate bacterial cells from the viral supernatant, and then carefully placed on the Biomek tablet. For each sample, two 250 ul aliquots are robotically distributed into two wells of a 96-well microtiter plate, and this process is repeated for each of the 48 samples until the entire 96 wells are filled. A solution of polyethylene glycol (PEG) then is added robotically to each well and mixed. The microtiter plate is covered with an acetate plate sealer, incubated at room temperature to precipitated the phage particles, and then centrifuged. The supernatant then is removed by inverting the plate and gently draining on a paper towel, without dislodging the pellet. After placing the microtiter plate back on the Biomek, a more dilute PEG solution is robotically added to each well. The plate then is covered with another sealer and centrifuged again. This rinse step aided in the removal of contaminating proteins and RNA. After removing the supernatant, as before, and placing the microtiter plate back on the Biomek, a Triton X-100 detergent solution is robotically added to each well. The plate is agitated gently and the sample from each pair of wells is robotically transferred to microcentrifuge tubes, which then are capped and placed in an 80deg C water bath for 10 minutes to aid in the detergent solubilization of phage coat proteins. After a brief centrifugation to collect condensation, the single-stranded DNA is ethanol precipitated, dried, and resuspended. An aliquot from each DNA sample is subjected to agarose gel electrophoresis to crudely assay concentration and purity. The yield of single-stranded template is approximately 2-3 ug per sample.

Protocol

The entire procedure will require 9 rows of P250 tips (counting from the center of the Biomek tablet towards the left) for the isolation of 48 templates (48ISOL). The reagent module should contain PEG-2000, Triton-Tris-EDTA, and ethanol-acetate, respectively.

1. Prepare an early log phase JM101 culture in 50 ml of 2xTY, as above.

2. Using sterile toothpicks, transfer individual M13 plaques into 12 X 75 mm Falcon tubes containing 1 ml early log phase cell cultures, and incubate for 4-6 hours at 37degC with shaking at 250 rpm. (Growth for longer than 6 hours results in cell lysis and contamination of the phage DNA by cellular proteins and nucleic acids).

3. Separate the bacterial cells from the viral-containing supernatant by centrifugation at 12,000 rpm for 15 minutes at 4degC. Carefully open the tubes and place on the Biomek tablet..

4. The Biomek will distribute two 250 ul aliquots of viral supernatant per sample into the wells of a 96-well flat-bottomed microtiter plate (Dynatech). The Biomek then will add 50 ul of 20% PEG/2.5 M NaCl solution to each well, and mix by pipetting up and down.

5. Cover the plate with an acetate plate sealer and incubate at room temperature for 15 minutes.

6. Pellet the precipitated phage by centrifuging the plate at 2400 rpm for 20 minutes in a Beckman GPR tabletop centrifuge. Remove the plate sealer and drain the PEG from the plate by gently draining upside down on a Kimwipe.

7. Return the plate to the tablet, and the Biomek will robotically add 200 ul of PEG:TE rinse solution to each well. Cover the plate with a plate sealer, centrifuge, and drain, as above.

8. Return the plate to the tablet, and the Biomek will add 70 ul of TTE solution to each well. Remove and gently agitate to resuspend.

9. The Biomek then will robotically pool the contents from each pair of wells into 1.5 ml microcentrifuge tubes.

10. Incubate the tubes at 80degC for 10 minutes to denature the viral protein coat and then centrifuge briefly to reclaim condensation.

11. Ethanol precipitate the DNA by adding 500 ul ethanol/acetate to each tube, as described above.

12. Resuspend the DNA templates in 20 ul of 10:0.1 TE buffer.